Small molecule stimulators of steroid receptor coactivator-3 and methods of their use as cardioprotective and/or vascular regenerative agents

ABSTRACT

Small molecule stimulators of steroid receptor coactivator-3 (SRC-3) and methods of their use as cardioprotective agents are provided. The small molecule stimulators are useful for promoting cardiac protection and repair and vascular regeneration after myocardial infarction. The compounds are also useful in preventing cardiac hypertrophy and collagen deposition and improving cardiac post-infarction function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/554,733, filed Aug. 29, 2019, which claims priority to U.S.Provisional Application Nos. 62/724,281, filed Aug. 29, 2018, and62/825,358, filed Mar. 28, 2019, which are incorporated herein byreference in their entireties.

BACKGROUND

A determinant of myocardial infarction (MI)-induced heart failure is aprogressive remodeling of cardiac tissue that is associated with loss ofmyocytes, inflammation, fibrosis, and a major depression of cardiacejection fraction. One promising therapeutic approach to improvingcardiac function is prevention of detrimental remodeling of cardiactissue in situ by directly preserving functional myocardium. A majorhurdle to maintaining cardiac function after infarction includes thetissue destruction and the adult heart's limited and restrictedregenerative potential, which poses a barrier to therapies designed topromote tissue reprogramming and repair.

SUMMARY

Described herein are small molecule stimulators of steroid receptorcoactivator-3 (SRC-3) and methods of their use as cardioprotectiveand/or vascular regenerative agents. The compounds described herein areuseful for promoting cardiac protection and repair and vascularregeneration after myocardial infarction. The methods includeadministering to a subject a compound as described herein.

Small molecule SRC-3 stimulators include compounds of the followingformula:

and pharmaceutically acceptable salts or prodrugs thereof. In thesecompounds, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are eachindependently selected from CR¹ and N, wherein each R¹ is hydrogen,halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstitutedC₁₋₆ alkyl; and R² is substituted or unsubstituted cycloalkyl orsubstituted or unsubstituted heterocycloalkyl. Optionally, the compoundhas the following formula:

wherein m and n are each independently 1, 2, 3, 4, or 5.

Optionally, the compound has the following formula:

wherein m and n are each independently 1, 2, 3, or 4.

In the compounds described herein, R² is optionally selected from thegroup consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Optionally, the compound is selected fromthe group consisting of:

Optionally, the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt or prodrug thereof.

Also described herein are methods for treating an ischemic injury (e.g.,a myocardial infarction or a stroke) in a subject, comprisingadministering to the subject an effective amount of a compound of thefollowing formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thecompounds for use in this method, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹,and A¹⁰ are each independently selected from CR¹ and N, wherein each R¹is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted orunsubstituted C₁₋₆ alkyl; and X is NR², CR³R⁴, or O, wherein R², R³, andR⁴ are each independently selected from the group consisting ofhydrogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted cycloalkyl, and substituted or unsubstitutedheterocycloalkyl. Optionally, the compound is selected from the groupconsisting of:

Optionally, the method can further comprise selecting a subject who hassuffered a myocardial infarction or who has suffered a stroke or othervascular impairments to the central nervous system.

Further described herein are methods of reducing a myocardial infarctsize in a subject who has suffered a myocardial infarction. The methodcan comprise administering to the subject an effective amount of acompound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thecompounds for use in the methods described herein, A¹, A², A³, A⁴, A⁵,A⁶, A⁷, A⁸, A⁹, and A¹⁰ are each independently selected from CR¹ and N,wherein each R¹ is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, orsubstituted or unsubstituted C₁₋₆ alkyl; and X is NR², CR³R⁴, or O,wherein R², R³, and R⁴ are each independently selected from the groupconsisting of hydrogen, substituted or unsubstituted C₁₋₆ alkyl,substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocycloalkyl. Optionally, the compound is selectedfrom the group consisting of:

Optionally, the myocardial infarct size is reduced by at least 5% (e.g.,by at least 15%) as compared to a myocardial infarct size in anuntreated subject who has suffered a myocardial infarction.

Also described herein are methods of preventing or reducingcardiomyocyte loss, improving cardiac vascular perfusion, and/orimproving central nervous system vascular perfusion in a subject who hassuffered a myocardial infarction or stroke, comprising administering tothe subject an effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thecompounds for use in this method, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹,and A¹⁰ are each independently selected from CR¹ and N, wherein each R¹is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted orunsubstituted C₁₋₆ alkyl; and X is NR², CR³R⁴, or O, wherein R², R³, andR⁴ are each independently selected from the group consisting ofhydrogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted cycloalkyl, and substituted or unsubstitutedheterocycloalkyl. Optionally, the compound is selected from the groupconsisting of:

Also described herein are methods for improving cardiovascular functionand/or central nervous system vascular function in a subject, comprisingadministering to the subject an effective amount of a compound of thefollowing formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thecompounds for use in this method, A, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, andA¹⁰ are each independently selected from CR¹ and N, wherein each R¹ ishydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted orunsubstituted C₁₋₆ alkyl; and X is NR², CR³R⁴, or O, wherein R², R³, andR⁴ are each independently selected from the group consisting ofhydrogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted cycloalkyl, and substituted or unsubstitutedheterocycloalkyl. Optionally, the compound is selected from the groupconsisting of:

Optionally, the subject has suffered an ischemic injury (e.g., amyocardial infarction or stroke). Optionally, the subject is an elderlysubject.

Also described herein are methods for promoting wound healing in asubject, comprising administering to the subject an effective amount ofa compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thecompounds for use in this method, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹,and A¹⁰ are each independently selected from CR¹ and N, wherein each R¹is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted orunsubstituted C₁₋₆ alkyl; and X is NR², CR³R⁴, or O, wherein R², R³, andR⁴ are each independently selected from the group consisting ofhydrogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted cycloalkyl, and substituted or unsubstitutedheterocycloalkyl. Optionally, the compound is selected from the groupconsisting of:

Optionally, the subject has suffered an ischemic injury (e.g., amyocardial infarction or stroke). Optionally, the subject is an elderlysubject.

Further described herein are methods for treating or preventinghypertrophic cardiomyopathy in a subject, comprising administering tothe subject an effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thecompounds for use in this method, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹,and A¹⁰ are each independently selected from CR and N, wherein each R¹is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted orunsubstituted C₁₋₆ alkyl; and X is NR², CR³R⁴, or O, wherein R², R³, andR⁴ are each independently selected from the group consisting ofhydrogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted cycloalkyl, and substituted or unsubstitutedheterocycloalkyl. Optionally, the compound is selected from the groupconsisting of:

Optionally, the subject has suffered an ischemic injury (e.g., amyocardial infarction or stroke).

The details of one or more embodiments are set forth in the drawings andthe description below. Other features, objects, and advantages will beapparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 contains graphs showing the expression of NCOA3 in normal humanhearts (left panel) and in muscle tissues (right panel).

FIG. 2 is an image of a heart from a mouse injected with adeno-SRC3before harvesting.

FIG. 3A depicts an experimental timeline for drug treatment andechocardiography measurements after myocardial infarction (MI). FIG. 3Bis a graph showing the heart weight and tibia length ratios (HW/TL) ofmice after a myocardial infarction. FIG. 3C is a graph showing theeffects of MCB-613 treatment in mice after a myocardial infarction. FIG.3D contains images of mouse hearts harvested after a myocardialinfarction and stained to visualize collagen fibers.

FIG. 4 is a graph showing the effects of Compound 10-1 treatment in miceafter a myocardial infarction.

FIG. 5A is a plot depicting a comprehensive single cell transcriptionalprofiling of non-myocyte cells in an adult mouse heart. FIG. 5B is agraph showing the different cell types present in an MCB-613 treatedheart after myocardial infarction. FIG. 5C is a Venn analysis of threecell clusters with endothelial signature.

FIG. 6A is a heat map showing the metabolomics for long-chain fattyacids in mouse hearts after a myocardial infarction. FIG. 6B is a heatmap showing the metabolomics for methylglutaryl carnitine in mousehearts after myocardial infarction.

FIG. 7, upper panel shows that MCB-613 selectively stimulates theintrinsic transcriptional activity of SRCs. FIG. 7, middle panel showsthat Compound 10-1 selectively stimulates the intrinsic transcriptionalactivity of SRCs. FIG. 7, bottom panel shows that Compound 10-2selectively stimulates the intrinsic transcriptional activity of SRCs.

FIG. 8 contains pictures of heart cross sections at the level of thepapillary muscle after myocardial infarction and after treatment withCompound 10-1.

FIG. 9 contains graphs showing the results of a progressive maximalexercise test in mice treated with saline (“saline”), mice treated withMCB-613 (“MCB-613), and non-infarcted wild-type mice (“WT”). The upperpanel shows the carbon dioxide expiration and the lower panel shows theoxygen consumption.

FIGS. 10A-10E show that MCB-613 stimulates angiogenesis in chicken eggsand in mouse hearts three days post-MI. For FIG. 10A, cardiacfibroblasts were treated with DMSO or MCB-613 for 24 hours. Totalprotein was then isolated and immunoblotted for SRC-1, -2 and -3. Hsp90was used a loading control. For FIG. 10B, cardiac fibroblasts weretransfected with a GAL4 DNA binding site-luciferase reporter (pG5-luc)and GAL4-DNA binding domain-full length SRC-1, -2 or -3 fusion(pBIND-SRC) or control pBIND expression vectors. Post transfection,cells were treated with DMSO or MCB-613 for 24 hours. Total protein wasisolated and measured for luciferase activity. Relative light units(RLU) were calculated by normalizing the luciferase activity to totalprotein concentration (n=3) (* P<0.05). For FIG. 10C, cardiacfibroblasts were treated with DMSO or MCB-613 for 24 hours and thenconditioned with endothelial growth media without drug for an additional24 hours. Conditioned cells were then plated in matrigel to allow tubeformation overnight and tubes were then stained with Calcein AM dye andimaged. For FIG. 10D, chicken eggs were treated with DMSO or MCB-613 andvessel area was measured at days one and three. Mouse embryonicfibroblasts (MEFs) were treated with dimethyl sulfoxide (DMSO) orMCB-613 for 24 hours then placed on a membrane in chicken eggs. Vesselarea was measured at days one and three. Data are presented as percentincrease over control for each condition. Six eggs were used for eachcondition * P<0.05. For FIG. 10E, mice were treated with MCB-613 orcontrol two hours post-MI. Hearts were fixed and immunostained forendothelial-cell-specific CD31. FIG. 10E shows representative images ofinfarct border zones from three control mice and three mice treated withMCB-613. FIG. 10E also contains a bar graph showing the quantificationCD31 immuno-stain density per area of tissue for two fields per borderzone * P<0.05.

FIGS. 11A-11G show that MCB-613 improves cardiac function followingmyocardial infarction. FIG. 11A is a schematic representation ofexperimental procedures. Mice were treated with MCB-613 or control twohours after permanent ligation of the left anterior descending coronaryartery and for six additional days and at weeks eight and 16 asindicated. For FIG. 11, ejection fraction was measured byechocardiography at the indicated times and hearts were harvested at 24hours and 12 weeks. * P<0.05. For FIG. 11C, heart weights were comparedto tibia length 12 weeks post-MI. FIG. 11D shows a representative imageof a center slice of a mouse heart in axial (short axis), coronal (longaxis), and sagittal views showing differences in morphology and ¹⁸F-FDGuptake between control (no MI), MI, and MI plus MCB-613 at two weekspost-MI. The arrow indicates the infarct zone. n=6 control no MI, n=6 MIplus vehicle control, n=4 MI plus MCB-613. For FIG. 11E, MCB-613-treatedhearts (n=2; infarct sizes 44% and 31%) and MCB-613-treated hearts at 12weeks (n=4; infarct sizes 22%, 3%, 20% and 14%) were fixed and stainedwith Picrosirus red. FIG. 11E also contains a bar graph showing thequantification of percent fibrosis at the border zones of each heart.Scale bars: 2000 μm and 20 μm. FIG. 11F shows representative electronmicrographs of border area 72 hours post-MI. My=myofibrils.Mi=mitochondria. Scale bar=1 μm. FIG. 11G, shows representative TUNELstaining from control and MCB-613 treated hearts 24 hours post-MI. n=4hearts per group. Scale bars: 2000 μm and 20 μm.

FIG. 12 contains graphs showing the results of a progressive maximalexercise test in non-infarcted wild-type mice treated with saline (“WTsaline”), non-infarcted wild-type mice treated with MCB-613 (“WTMCB-613”), mice treated with saline post-MI (“MI saline”), and micetreated with MCB-613 post-MI (“MI MCB-613”). The left panel shows theoxygen consumption and the right panel shows the carbon dioxideexpiration.

FIGS. 13A-13F shows RNA transcriptional profiling of cardiomyocytes andsingle cell analysis of interstitial cells 12 weeks post-MI that revealsthat the MCB-613 protective response is associated with improvedoxidative phosphorylation, decreased inflammation, and decreased immunecells. FIG. 13A is a schematic representation of isolation procedures toobtain cardiomyocytes for total RNA-sequencing and non-cardiomyocytesfor single-cell RNA-seq analysis from control treated and MCB-613treated mice 12 weeks post-MI. n=2 hearts/group.

FIG. 13B is a heat map analysis of genes identified by RNA-seq anddifferentially expressed in cardiomyocytes from two mice treated withMCB-613 versus two mice treated with saline 10 weeks post-MI. FIG. 13Cis a gene set enrichment analysis of upregulated and downregulated genesin cardiomyocytes of MCB-613 versus control treated hearts. FIG. 13Ddepicts cell populations identified by unsupervised clustering. Each dotindicates a single cell. FIG. 13E is a heat map that indicatesestablished cell type markers used to specifically identify eachcluster. FIG. 13F shows representative TUNEL staining from control andMCB-613 treated hearts 24 hours post-MI. n=4 hearts per group. Scalebars: 2000 μm and 20 μm.

FIG. 14A is a Venn analysis of fibroblasts cluster gene expression. FIG.14B is a Venn analysis of endothelial cluster gene expression. FIG. 14Cis a Venn analysis of macrophage gene expression. FIG. 14D is a gene setenrichment analysis of upregulated and downregulated genes ingranulocytes of MCB-613 over control-treated hearts.

FIGS. 15A-15D show that MCB-613 regulates sustained immune andendothelial cell responses 12 weeks post-MI. FIG. 15A shows the numberof up- and down-regulated genes in non-myocyte cells from control micecompared to MCB-613 treated mice. FIG. 15B contains a receptor-ligandanalysis of intercellular communication between cardiac cell typesexcluding cardiomyocytes. The lines indicate communication between thetwo cell types. The directionality of the ligand-receptor pairing beginsat the node and ends at the cognate receptor as illustrated in thefigure legend. The thickness of the line reflects the number ofligand-receptor pairings. The loops represent autocrine signalingcircuits. FIG. 15C is a heat map of ligand-receptor pairings betweengranulocytes, fibroblast clusters and macrophage C₄. FIG. 15D is a heatmap showing top 50 up and down-regulated drug-responsive genes for 277and 310 granulocytes from control and MCB-613-treated hearts,respectively.

FIGS. 16A-16C show that MCB-613 decreases B lymphocytes and monocytesand upregulates granulocyte genes and lysozyme as early as 24 hourspost-MI. FIG. 16A shows the quantification of cardiac immune cells byfluorescence-activated cell sorting (FACS) immune phenotyping analysis24 hours post-MI from control and MCB-613 treated mice. FIG. 16B showsmRNA expression in granulocytes and neutrophils isolated from bonemarrow 24 hours post MI and MCB 613 treatment. Total RNA was isolatedfrom neutrophil enriched and neutrophil depleted fractions of bonemarrow and converted to cDNA. Gene expression changes in S100a9, Tlr7and Lcn2 were measured by qPCR and 18s RNA expression was used as acontrol. N=6 each group * P<0.05. FIG. 16C shows representative LYZstaining from control and MCB-613 treated hearts 24 hours after MI. Thetop panels of FIG. 16C show a low magnification from endocardium toepicardium and the bottom panels show a high magnification ofsub-endocardial regions. The arrows indicate LYZ+ cells. The bar graphin FIG. 16C shows the quantification of LV density of LYZ+ cells. n=3hearts/group, >10 mm² imaged/heart, 24 hours after MI surgery * P<0.039.

FIG. 17 contains graphical representations of pharmacokinetics dataobtained for MCB-613, Compound 1, and Compound 2 in CD-1 mice.

DETAILED DESCRIPTION

Described herein are stimulators of steroid receptor coactivator (SRC)proteins and methods for their use. Steroid receptor coactivators aremembers of the p160 family of nuclear receptor coactivators and includeSRC-1, SRC-2 (TIF2/GRIP1), and SRC-3 (AIB1/RAC3/ACTR/pCIP). The smallmolecules described herein are stimulators of SRC-3 and are useful ascardioprotective and/or vascular regenerative agents. In particular, thecompounds are useful for promoting cardiac protection and repair andvascular regeneration after myocardial infarction or stroke. Thecompounds are also useful in preventing cardiac hypertrophy and collagendeposition and improving cardiac post-infarction function. The compoundshave been demonstrated to increase angiogenesis, increase vascularperfusion in the heart and central nervous system, and promote cardiacbeta oxidation. Administration of the compounds described herein alsosignificantly decreases the presence of methylglutaryl carnitine, ametabolite associated with dilated cardiomyopathy.

I. Compounds

A class of SRC stimulators described herein is represented by Formula I

and pharmaceutically acceptable salts or prodrugs thereof.

In Formula I, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are eachindependently selected from CR¹ and N. Each R¹ group present in FormulaI is independently selected from hydrogen, halogen, alkoxy, cyano,trifluoromethyl, and substituted or unsubstituted C₁₋₆ alkyl.

Also, in Formula I, X is NR², CR³R⁴, or O, wherein R², R³, and R⁴ areeach independently selected from the group consisting of hydrogen,substituted or unsubstituted C₁₋₆ alkyl, substituted or unsubstitutedcycloalkyl, and substituted or unsubstituted heterocycloalkyl.

As used herein, the terms alkyl, alkenyl, and alkynyl include straight-and branched-chain monovalent substituents. Examples include methyl,ethyl, isobutyl, 3-butynyl, and the like. Ranges of these groups usefulwith the compounds and methods described herein include C₁-C₂₀ alkyl,C₂-C₂₀ alkenyl, and C₂-C₂₀ alkynyl. Additional ranges of these groupsuseful with the compounds and methods described herein include C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₄ alkyl, C₂-C₄ alkenyl, and C₂-C₄ alkynyl.

Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly asalkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms orcombinations thereof within the backbone. Ranges of these groups usefulwith the compounds and methods described herein include C₁-C₂₀heteroalkyl, C₂-C₂₀ heteroalkenyl, and C₂-C₂₀ heteroalkynyl. Additionalranges of these groups useful with the compounds and methods describedherein include C₁-C₁₂ heteroalkyl, C₂-C₁₂ heteroalkenyl, C₂-C₁₂heteroalkynyl, C₁-C₆ heteroalkyl, C₂-C₆ heteroalkenyl, C₂-C₆heteroalkynyl, C₁-C₄ heteroalkyl, C₂-C₄ heteroalkenyl, and C₂-C₄heteroalkynyl.

The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclicalkyl groups having a single cyclic ring or multiple condensed rings.Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. Rangesof these groups useful with the compounds and methods described hereininclude C₃-C₂₀ cycloalkyl, C₃-C₂₀ cycloalkenyl, and C₃-C₂₀ cycloalkynyl.Additional ranges of these groups useful with the compounds and methodsdescribed herein include C₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloalkenyl, C₅-C₁₂cycloalkynyl, C₅-C₆ cycloalkyl, C₅-C₆ cycloalkenyl, and C₅-C₆cycloalkynyl.

The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynylare defined similarly as cycloalkyl, cycloalkenyl, and cycloalkynyl, butcan contain O, S, or N heteroatoms or combinations thereof within thecyclic backbone. Ranges of these groups useful with the compounds andmethods described herein include C₃-C₂₀ heterocycloalkyl, C₃-C₂₀heterocycloalkenyl, and C₃-C₂₀ heterocycloalkynyl. Additional ranges ofthese groups useful with the compounds and methods described hereininclude C₅-C₁₂ heterocycloalkyl, C₅-C₁₂ heterocycloalkenyl, C₅-C₁₂heterocycloalkynyl, C₅-C₆ heterocycloalkyl, C₅-C₆ heterocycloalkenyl,and C₅-C₆ heterocycloalkynyl.

Aryl molecules include, for example, cyclic hydrocarbons thatincorporate one or more planar sets of, typically, six carbon atoms thatare connected by delocalized electrons numbering the same as if theyconsisted of alternating single and double covalent bonds. An example ofan aryl molecule is benzene. Heteroaryl molecules include substitutionsalong their main cyclic chain of atoms such as O, N, or S. Whenheteroatoms are introduced, a set of five atoms, e.g., four carbon and aheteroatom, can create an aromatic system. Examples of heteroarylmolecules include furan, pyrrole, thiophene, imadazole, oxazole,pyridine, and pyrazine. Aryl and heteroaryl molecules can also includeadditional fused rings, for example, benzofuran, indole, benzothiophene,naphthalene, anthracene, and quinoline. The aryl and heteroarylmolecules can be attached at any position on the ring, unless otherwisenoted.

The term alkoxy as used herein is an alkyl group bound through a single,terminal ether linkage. Likewise, the term aryloxy as used herein is anaryl group bound through a single, terminal ether linkage.

The term hydroxyl as used herein is represented by the formula —OH.

The terms amine or amino as used herein are represented by the formula—NZ¹Z², where Z¹ and Z² can each be a substitution group as describedherein, such as hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl,aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, orheterocycloalkenyl group described above.

The alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl,heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, orheterocycloalkyl molecules used herein can be substituted orunsubstituted. As used herein, the term substituted includes theaddition of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, orheterocycloalkyl group to a position attached to the main chain of thealkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl,heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, orheterocycloalkyl, e.g., the replacement of a hydrogen by one of thesemolecules. Examples of substitution groups include, but are not limitedto, hydroxyl, halogen (e.g., F, Br, Cl, or I), and carboxyl groups.Conversely, as used herein, the term unsubstituted indicates the alkoxy,aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl,heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, orheterocycloalkyl has a full complement of hydrogens, i.e., commensuratewith its saturation level, with no substitutions, e.g., linear decane(—(CH₂)₉—CH₃).

In some examples, Formula I is represented by Structure I-A:

In Structure I-A, A, A², A³, A⁴, A⁵, A⁶, A⁷, A, A⁹, A¹⁰, and R² are asdefined above for Formula I. In some examples of Structure I-A, each ofA¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are CR¹, where each R isindependently selected from a group as defined above for Formula I. Forexample, the compound of Structure I-A can be represented by StructureI-A1:

In Structure I-A1, m and n are each independently 1, 2, 3, 4, or 5. Inother words, the phenyl rings of the molecule can include from one tofive R groups. Each of the R groups can be independently selected from agroup as defined above for Formula I.

In some examples of Structure I-A, one or more of A, A², A³, A⁴, A⁵, A⁶,A⁷, A⁸, A⁹, and A¹⁰ can be N. For example, the compound of Structure I-Acan be represented by Structure I-A2 Structure I-A3, or Structure I-A4:

In Structure I-A2, Structure I-A3, and Structure I-A4, m and n are eachindependently 1, 2, 3, or 4. In other words, the phenyl rings of themolecule can include from one to four R¹ groups. Each of the R¹ groupscan be independently selected from a group as defined above for FormulaI.

Optionally, in Structure I-A1, Structure I-A2, Structure I-A3, and/orStructure I-A4, R² is substituted or unsubstituted cycloalkyl orsubstituted or unsubstituted heterocycloalkyl. In some examples, R² isselected from the group consisting of cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

In some examples, Formula I is represented by Structure I-B:

In Structure I-B, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, A¹⁰, R³, and R⁴are as defined above for Formula I. In some examples of Structure I-B,each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are CR¹, where eachR¹ is independently selected from a group as defined above for FormulaI. For example, the compound of Structure I-B can be represented byStructure I-B1:

In Structure I-B1, m and n are each independently 1, 2, 3, 4, or 5. Inother words, the phenyl rings of the molecule can each independentlyinclude from one to five R¹ groups. Each of the R¹ groups can beindependently selected from a group as defined above for Formula I.

In some examples of Structure I-B, one or more of A¹, A², A³, A⁴, A⁵,A⁶, A⁷, A⁸, A⁹, and A¹⁰ can be N. For example, the compound of StructureI-B can be represented by Structure I-B2, Structure I-B3, or StructureI-B4:

In Structure I-B2, Structure I-B3, and Structure I-B4, m and n are eachindependently 1, 2, 3, or 4. In other words, the phenyl rings of themolecule can each independently include from one to four R¹ groups. Eachof the R¹ groups can be independently selected from a group as definedabove for Formula I.

In some examples, Formula I is represented by Structure I-C:

In Structure I-C, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are asdefined above for Formula I. In some examples of Structure I-C, each ofA¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are CR¹, where each R¹ isindependently selected from a group as defined above for Formula I. Forexample, the compound of Structure I-C can be represented by StructureI-C1:

In Structure I-C₁, m and n are each independently 1, 2, 3, or 4. Inother words, the phenyl rings of the molecule can each independentlyinclude from one to four R¹ groups. Each of the R¹ groups can beindependently selected from a group as defined above for Formula I.

In some examples of Structure I-C, one or more of A¹, A², A³, A⁴, A⁵,A⁶, A⁷, A⁸, A⁹, and A¹⁰ can be N. For example, the compound of StructureI-C can be represented by Structure I-C2, Structure I-C3, or StructureI-C4:

In Structure I-C2, Structure I-C3, and Structure I-C4, m and n are eachindependently 1, 2, 3, or 4. In other words, the phenyl rings of themolecule can each independently include from one to four R¹ groups. Eachof the R¹ groups can be independently selected from a group as definedabove for Formula I.

Examples of Formula I include the following compounds:

In some embodiments, the compound is SYC-944 (Compound 2-8) (alsoreferred to herein as MCB-613). In some embodiments, the compound is notSYC-944 (Compound 2-8) (also referred to herein as MCB-613). In someembodiments, the compound is Compound 9-2, Compound 9-8, Compound 10-1,or Compound 10-2.

II. Methods of Making the Compounds

The compounds described herein can be prepared in a variety of ways. Thecompounds can be synthesized using various synthetic methods. At leastsome of these methods are known in the art of synthetic organicchemistry. The compounds described herein can be prepared from readilyavailable starting materials. Optimum reaction conditions can vary withthe particular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Variations on Formula I include the addition, subtraction, or movementof the various constituents as described for each compound. Similarly,when one or more chiral centers are present in a molecule, all possiblechiral variants are included. Additionally, compound synthesis caninvolve the protection and deprotection of various chemical groups. Theuse of protection and deprotection, and the selection of appropriateprotecting groups can be determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Wuts,Greene's Protective Groups in Organic Synthesis, 5th. Ed., Wiley & Sons,2014, which is incorporated herein by reference in its entirety.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H-NMR or ¹³C-NMR),infrared spectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Exemplary methods for synthesizing compounds as described herein areprovided in Example 1 below and in International Patent ApplicationPublication No. WO 2016/109470, which is incorporated herein byreference.

III. Pharmaceutical Formulations

The compounds described herein or derivatives thereof can be provided ina pharmaceutical composition. Depending on the intended mode ofadministration, the pharmaceutical composition can be in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include a therapeuticallyeffective amount of the compound described herein or derivatives thereofin combination with a pharmaceutically acceptable carrier and, inaddition, may include other medicinal agents, pharmaceutical agents,carriers, or diluents. By pharmaceutically acceptable is meant amaterial that is not biologically or otherwise undesirable, which can beadministered to an individual along with the selected compound withoutcausing unacceptable biological effects or interacting in a deleteriousmanner with the other components of the pharmaceutical composition inwhich it is contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005.Examples of physiologically acceptable carriers include buffers, such asphosphate buffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers, such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates, including glucose, mannose, or dextrins; chelatingagents, such as EDTA; sugar alcohols, such as mannitol or sorbitol;salt-forming counterions, such as sodium; and/or nonionic surfactants,such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.).

Compositions containing the compound described herein or derivativesthereof suitable for parenteral injection may comprise physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

These compositions may also contain adjuvants, such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be promoted by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Isotonic agents, for example, sugars, sodium chloride, and thelike may also be included. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds describedherein or derivatives thereof include capsules, tablets, pills, powders,and granules. In such solid dosage forms, the compounds described hereinor derivatives thereof is admixed with at least one inert customaryexcipient (or carrier), such as sodium citrate or dicalcium phosphate,or (a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They may contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration of the compounds describedherein or derivatives thereof include pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage forms may contain inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents, and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils,in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include additionalagents, such as wetting, emulsifying, suspending, sweetening, flavoring,or perfuming agents.

Suspensions, in addition to the active compounds, may contain additionalagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions of the compounds described herein or derivatives thereoffor rectal administrations are optionally suppositories, which can beprepared by mixing the compounds with suitable non-irritating excipientsor carriers, such as cocoa butter, polyethyleneglycol or a suppositorywax, which are solid at ordinary temperatures but liquid at bodytemperature and, therefore, melt in the rectum or vaginal cavity andrelease the active component.

Dosage forms for topical administration of the compounds describedherein or derivatives thereof include ointments, powders, sprays, andinhalants. The compounds described herein or derivatives thereof areadmixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, ointments, powders, and solutions arealso contemplated as being within the scope of the compositions.

The compositions can include one or more of the compounds describedherein and a pharmaceutically acceptable carrier. As used herein, theterm pharmaceutically acceptable salt refers to those salts of thecompound described herein or derivatives thereof that are, within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use, as well as the zwitterionicforms, where possible, of the compounds described herein. The term saltsrefers to the relatively non-toxic, inorganic and organic acid additionsalts of the compounds described herein. These salts can be prepared insitu during the isolation and purification of the compounds or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonatesalts, and the like. These may include cations based on the alkali andalkaline earth metals, such as sodium, lithium, potassium, calcium,magnesium, and the like, as well as non-toxic ammonium, quaternaryammonium, and amine cations including, but not limited to ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, ethylamine, and the like. (See S. M.Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated hereinby reference in its entirety, at least, for compositions taughttherein.)

Administration of the compounds and compositions described herein orpharmaceutically acceptable salts thereof can be carried out usingtherapeutically effective amounts of the compounds and compositionsdescribed herein or pharmaceutically acceptable salts thereof asdescribed herein for periods of time effective to treat a disorder. Theeffective amount of the compounds and compositions described herein orpharmaceutically acceptable salts thereof as described herein may bedetermined by one of ordinary skill in the art and includes exemplarydosage amounts for a mammal of from about 0.5 to about 200 mg/kg of bodyweight of active compound per day, which may be administered in a singledose or in the form of individual divided doses, such as from 1 to 4times per day. Alternatively, the dosage amount can be from about 0.5 toabout 150 mg/kg of body weight of active compound per day, about 0.5 to100 mg/kg of body weight of active compound per day, about 0.5 to about75 mg/kg of body weight of active compound per day, about 0.5 to about50 mg/kg of body weight of active compound per day, about 0.01 to about50 mg/kg of body weight of active compound per day, about 0.05 to about25 mg/kg of body weight of active compound per day, about 0.1 to about25 mg/kg of body weight of active compound per day, about 0.5 to about25 mg/kg of body weight of active compound per day, about 1 to about 20mg/kg of body weight of active compound per day, about 1 to about 10mg/kg of body weight of active compound per day, about 20 mg/kg of bodyweight of active compound per day, about 10 mg/kg of body weight ofactive compound per day, about 5 mg/kg of body weight of active compoundper day, about 2.5 mg/kg of body weight of active compound per day,about 1.0 mg/kg of body weight of active compound per day, or about 0.5mg/kg of body weight of active compound per day, or any range derivabletherein. Optionally, the dosage amounts are from about 0.01 mg/kg toabout 10 mg/kg of body weight of active compound per day. Optionally,the dosage amount is from about 0.01 mg/kg to about 5 mg/kg. Optionally,the dosage amount is from about 0.01 mg/kg to about 2.5 mg/kg.

Those of skill in the art will understand that the specific dose leveland frequency of dosage for any particular subject may be varied andwill depend upon a variety of factors, including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the species, age, body weight, general health, sex anddiet of the subject, the mode and time of administration, rate ofexcretion, drug combination, and severity of the particular condition.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the disease ordisorder, and should be decided according to the judgment of thepractitioner and each subject's circumstances. Effective doses can beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. Further, depending on the route of administration,one of skill in the art would know how to determine doses that result ina plasma concentration for a desired level of response in the cells,tissues and/or organs of a subject.

IV. Methods of Use

Provided herein are methods to treat a myocardial infarction or otherischemic injury (e.g., a stroke) in a subject. The methods includeadministering to a subject an effective amount of one or more of thecompounds or compositions described herein, or a pharmaceuticallyacceptable salt or prodrug thereof. Effective amount, when used todescribe an amount of compound in a method, refers to the amount of acompound that achieves the desired pharmacological effect or otherbiological effect.

Also contemplated is a method that includes administering to the subjectan amount of one or more compounds described herein such that an in vivoconcentration at a target cell in the subject corresponding to theconcentration administered in vitro is achieved.

Further described herein are methods for reducing a myocardial infarctsize in a subject who has suffered a myocardial infarction. The methodsinclude administering to the subject an effective amount of one or morecompounds or a composition as described herein. The myocardial infarctsize can be reduced by at least 5% as compared to a myocardial infarctsize in an untreated subject who has suffered a myocardial infarction(e.g., a subject who has suffered a myocardial infarction and has notbeen administered any treatment for the myocardial infarction or asubject who has suffered a myocardial infarction and has beenadministered a therapeutic agent other than a compound or composition asdescribed herein). Optionally, the myocardial infarct size can bereduced by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% as compared to amyocardial infarct size in an untreated subject who has suffered amyocardial infarction. The compounds and compositions described hereinare also useful in preventing or reducing cardiomyocyte loss in asubject who has suffered a myocardial infarction. The methods forpreventing or reducing cardiomyocyte loss in a subject who has suffereda myocardial infarction include administering to the subject aneffective amount of one or more compounds or a composition as describedherein.

Further described herein are methods for improving cardiovascularfunction, improving cardiac vascular perfusion, improving centralnervous system vascular function, improving central nervous systemvascular perfusion, promoting wound healing, and/or preventing ortreating hypertrophic cardiomyopathy in a subject. The methods includeadministering to the subject an effective amount of one or morecompounds or a composition as described herein. Optionally, the subjecthas suffered an ischemic injury, such as a myocardial infarction orstroke. Optionally, the subject is an elderly individual, an obeseindividual, a diabetic individual, an individual suffering from ametabolic syndrome, an individual exposed to smoke (e.g., smokers andindividuals exposed to second-hand smoke for an extended period), anindividual suffering from elevated blood pressure, blood cholesterol, ortriglyceride levels, or an individual suffering from an autoimmunecondition.

The methods for treating a myocardial infarction, reducing a myocardialinfarct size, preventing or reducing cardiomyocyte loss, improvingcardiovascular function, improving cardiac vascular perfusion, improvingcentral nervous system vascular function, improving central nervoussystem vascular perfusion, promoting wound healing, and/or preventing ortreating hypertrophic cardiomyopathy in a subject can further compriseadministering to the subject one or more additional agents. The one ormore additional agents and the compounds described herein orpharmaceutically acceptable salts or prodrugs thereof can beadministered in any order, including concomitant, simultaneous, orsequential administration. Sequential administration can beadministration in a temporally spaced order of up to several days apart.The methods can also include more than a single administration of theone or more additional agents and/or the compounds described herein orpharmaceutically acceptable salts or prodrugs thereof. Theadministration of the one or more additional agents and the compoundsdescribed herein or pharmaceutically acceptable salts or prodrugsthereof can be by the same or different routes and concurrently orsequentially.

Additional therapeutic agents include, but are not limited to,antiplatelet agents, statins, beta blockers, andrenin-angiotensin-aldosterone system (RAAS) blockers (e.g.,angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptorblockers (ARBs)). Illustrative, non-limiting examples of antiplateletagents useful as an additional therapeutic agent as described hereininclude glycoprotein (GP) IIb/IIIa receptor antagonists (e.g.,abciximab, eptifibatide, and tirofiban), dipyridamole, cylooxygenaseinhibitors (e.g., acetylsalicylic acid, ibuprofen indomethacin, andsulfinpyrazone), adenosine diphosphate (ADP) receptor antagonists (e.g.,clopidogrel and ticlopidine), and phosphodiesterase inhibitors (e.g.,cilostazol).

Illustrative, non-limiting examples of statins useful as an additionaltherapeutic agent as described herein include atorvastatin,cerivastatin, pravastatin, lovastatin, mevastatin, simvastatin,rosuvastatin, fluvastatin, and pitavastatin.

Illustrative examples of beta blockers useful as an additionaltherapeutic agent as described herein include, but are not limited to,acebutolol, atenolol, betaxolol, bisoprolol fumarate, carteolol,carvedilol, esmolol, labetalol, metoprolol, nadolol, nebivolol,penbutolol, pindolol, propranolol, sotalol, and timolol.

Illustrative examples of renin-angiotensin-aldosterone system (RAAS)blockers (e.g., angiotensin-converting enzyme (ACE) inhibitors andangiotensin receptor blockers (ARBs)) useful as an additionaltherapeutic agent as described herein include, but are not limited to,aliskiren, enalkiren, remikirem, benazepril, benazeprilat, captopril,enalapril, lisinopril, perindopril, quinapril, ramipril, trandolapril,fosinopril, moexipril, perindopril, losartan, valsartan, irbesartan,candesartan, telmisartan, tasosartan, eprosartan, spironolactone, andeplerenone.

Any of the aforementioned therapeutic agents can be used in anycombination with the compositions described herein. Combinations areadministered either concomitantly (e.g., as an admixture), separatelybut simultaneously (e.g., via separate intravenous lines into the samesubject), or sequentially (e.g., one of the compounds or agents is givenfirst followed by the second). Thus, the term combination is used torefer to concomitant, simultaneous, or sequential administration of twoor more agents.

Optionally, a compound or therapeutic agent as described herein may beadministered in combination with a surgery (e.g., a coronary arterybypass surgery), an angioplasty, a stenting, or another implantationprocedure.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. For prophylactic use, atherapeutically effective amount of the compounds and compositions orpharmaceutically acceptable salts thereof as described herein areadministered to a subject prior to onset (e.g., before obvious signs ofa myocardial infarction), during early onset (e.g., upon initial signsand symptoms of a myocardial infarction), or after the occurrence of amyocardial infarction. Prophylactic administration can occur for severaldays to years prior to the manifestation of symptoms of a myocardialinfarction. Therapeutic treatment involves administering to a subject atherapeutically effective amount of the compounds and compositions orpharmaceutically acceptable salts thereof as described herein after theoccurrence of a myocardial infarction.

The methods herein for prophylactic and therapeutic treatment optionallycomprise selecting a subject who has suffered or is at an elevated riskof suffering an ischemic injury such as a myocardial infarction orstroke (e.g., an obese individual, an elderly individual, or anindividual who has previously suffered an ischemic injury such as amyocardial infarction or stroke). A skilled artisan can make such adetermination using, for example, a variety of prognostic and diagnosticmethods, including, for example, a personal or family history of thedisease or condition, clinical tests (e.g., genetic tests), and thelike. Optionally, the methods herein can be used for preventing asubject who has suffered a myocardial infarction from suffering asubsequent myocardial infarction.

The compounds and compositions described herein or pharmaceuticallyacceptable salts thereof are useful for treating a myocardialinfarction, reducing a myocardial infarct size, preventing or reducingcardiomyocyte loss, improving cardiovascular function, promoting woundhealing, and/or preventing or treating hypertrophic cardiomyopathy inhumans, including, without limitation, pediatric and geriatricpopulations, and in animals, e.g., veterinary applications.

V. Kits

Also provided herein are kits for treating a myocardial infarction,reducing a myocardial infarct size, preventing or reducing cardiomyocyteloss, improving cardiovascular function, promoting wound healing, and/orpreventing or treating hypertrophic cardiomyopathy in a subject. A kitcan include any of the compounds or compositions described herein. Forexample, a kit can include one or more compounds of Formula I. A kit canfurther include one or more additional agents, such as antiplateletagents, statins, beta blockers, renin-angiotensin-aldosterone system(RAAS) blockers (e.g., angiotensin-converting enzyme (ACE) inhibitorsand angiotensin receptor blockers (ARBs)), and combinations of these.

A kit can include an oral formulation of any of the compounds orcompositions described herein. A kit can include an intravenous orintraperitoneal formulation of any of the compounds or compositionsdescribed herein. A kit can additionally include directions for use ofthe kit (e.g., instructions for treating a subject), a container, ameans for administering the compounds or compositions (e.g., a syringe),and/or a carrier.

As used herein the terms treatment, treat, or treating refer to a methodof reducing one or more symptoms of a disease or condition. Thus in thedisclosed method, treatment can refer to a 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% reduction in the severity of one or moresymptoms of the disease or condition. For example, a method for treatinga disease is considered to be a treatment if there is a 5% reduction inone or more symptoms or signs (e.g., a size of a myocardial infarct) ofthe disease in a subject as compared to a control. As used herein,control refers to the untreated condition (e.g., a myocardial infarctsize in an untreated subject who has suffered a myocardial infarction).Thus the reduction can be a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, or any percent reduction in between 5% and 100% as comparedto native or control levels. It is understood that treatment does notnecessarily refer to a cure or complete ablation of the disease,condition, or symptoms of the disease or condition.

As used herein, the terms prevent, preventing, and prevention of adisease or disorder refer to an action, for example, administration of acomposition or therapeutic agent, that occurs before or at about thesame time a subject begins to show one or more symptoms of the diseaseor disorder, which inhibits or delays onset or severity of one or moresymptoms of the disease or disorder.

As used herein, references to decreasing, reducing, or inhibitinginclude a change of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater as compared to a control level. Such terms can include, but donot necessarily include, complete elimination.

As used herein, subject means both mammals and non-mammals. Mammalsinclude, for example, humans; non-human primates, e.g., apes andmonkeys; cattle; horses; sheep; rats; mice; pigs; and goats. Non-mammalsinclude, for example, fish and birds.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application.

The examples below are intended to further illustrate certain aspects ofthe methods and compositions described herein, and are not intended tolimit the scope of the claims.

EXAMPLES Example 1: Synthesis of Compounds

All chemicals for synthesis were purchased from Alfa Aesar (Ward Hill,Mass.) or Aldrich (Milwaukee, Wis.). The compound identity wascharacterized by ¹H NMR on a Varian (Palo Alto, Calif.) 400-MRspectrometer. The purities of synthesized compounds were determined by aShimadzu Prominence HPLC with a Zorbax C18 (or C8) column (4.6×250 mm)monitored by UV at 254 nm. The purities of the reported compounds werefound to be >95%.

Synthesis of 1-cyclopropylpiperidin-4-one

Cyclopropylamine (6.9 mL, 100 mmol) and ethyl acrylate (22.3 mL, 210mmol, 2.1 equiv) were dissolved in absolute ethanol (50 mL). The mixturewas stirred at room temperature (rt) for 4 days. The volatiles wereremoved in vacuo to afford a crude oil, which was purified by columnchromatography (silica gel, n-hexanes:ethyl acetate from 10:1 to 1:1) togive diethyl 3,3′-(cyclopropylazanediyl)dipropanoate as a colorlessliquid (15.68 g, 61%).

Sodium hydride (60% dispersion in oil, 3.0 g, 75 mmol, 1.5 equiv) andtetrahydrofuran (THF, 30 mL) were placed in an oven-dried flask, towhich a solution of diethyl 3,3′-(cyclopropylazanediyl)dipropanoate(12.8 g, 50 mmol) in THE (20 mL) was added dropwise. Then absoluteethanol (2.9 mL, 50 mmol, 1.0 equiv) was added and the resulting mixturewas stirred at reflux for 24 hours. The reaction was quenched withsaturated ammonium chloride (50 mL). The mixture was extracted withdiethyl ether (3×100 mL) and the combined organic layers were washedwith water and brine, dried over Na₂SO₄. The volatiles were removed invacuo to afford a crude oil, which was purified by column chromatography(silica gel, n-hexanes:ethyl acetate from 10:1 to 2:1) to give1-cyclopropylpiperidin-4-one as a colorless liquid (4.52 g, 65% yield).¹H NMR (400 MHz, CDCl₃): δ 2.92 (t, J=6.2 Hz, 4H), 2.42 (t, J=6.2 Hz,4H), 1.80-1.70 (m, 1H), and 0.55-0.47 (m, 4H).

Synthesis of 1-isopentylpiperidin-4-one

Piperidin-4-one hydrochloride (13.56 g, 100 mmol),1-bromo-3-methylbutane (14.4 mL, 120 mmol, 1.2 equiv) and potassiumcarbonate (27.6 g, 200 mmol, 2.0 equiv) were dissolved in anacetonitrile/water (50/50 mL) mix solvent. The mixture was stirred atreflux for 12 hours. The mixture was cooled to rt and extracted withdiethyl ether (3×100 mL) and the combined organic layers were washedwith water and brine, and dried over Na₂SO₄. The volatiles were removedin vacuo to afford a crude oil, which was purified by columnchromatography (silica gel, n-hexanes:ethyl acetate from 10:1 to 2:1) togive 1-isopentylpiperidin-4-one as a colorless liquid (10.32 g, 61%yield). ¹H NMR (400 MHz, CDCl₃): δ 2.71 (t, J=5.9 Hz, 4H), 2.43 (t,J=5.9 Hz, 6H), 1.61 (septet, J=6.6 Hz, 1H), 1.40 (td, J=7.6, 6.6 Hz,2H), and 0.90 (d, J=6.6 Hz, 6H).

General Method for Synthesis of α,β-Unsaturated Ketone

N-protected piperidin-4-one (5 mmol) and aldehyde (11 mmol, 2.2 equiv)were dissolved in acetic acid (10 mL) to which concentrated hydrochloricacid (3 mL) was added. The mixture was stirred at rt for 12 hours. Thereaction was carefully quenched by saturated sodium bicarbonate. Themixture was extracted with ethyl acetate (3×30 mL) and the combinedorganic layers were washed with water and brine, and dried over Na₂SO₄.The volatiles were removed in vacuo to afford a crude oil, which waspurified by column chromatography (silica gel, n-hexanes:ethyl acetatefrom 5:1 to 1:1) to give α,β-unsaturated ketone (60-76% yield). Thecompounds described herein were prepared according to the generalmethod, including Compounds 9-2, 9-8, 10-1, and 10-2. Thecharacterizations for each of these compounds is provided below.

(3E,5E)-1-Cyclopropyl-3,5-bis(3-methoxybenzylidene)piperidin-4-one (9-2)

Yellow powder. ¹H NMR (400 MHz, CDCl₃): δ 7.75 (s, 2H), 7.36 (t, J=8.0Hz, 2H), 7.03 (d, J=8.0 Hz, 2H), 6.96 (s, 2H), 6.93 (d, J=8.0 Hz, 2H),3.99 (s, 4H), 3.85 (s, 6H), 1.96-1.91 (m, 1H), 0.51-0.47 (m, 2H), and0.41-0.38 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): 187.6, 159.7, 136.7, 136.4,133.8, 129.7, 123.0, 116.1, 114.7, 54.0, 51.3, 38.1, and 6.9; MS (ESI)[M+H]⁺ 376.5.

(3E,5E)-1-cyclopropyl-3,5-bis(pyridin-3-ylmethylene)piperidin-4-one(9-8)

Orange powder. ¹H NMR (400 MHz, CDCl₃): δ 8.67 (s, 2H), 8.59 (d, J=4.8Hz, 2H), 7.73 (s, 2H), 7.70 (d, J=8.0 Hz, 2H), 7.37 (dd, J=8.0, 4.8 Hz,2H), 3.99 (s, 4H), 1.99-1.94 (m, 1H), 0.58-0.51 (m, 2H), and 0.47-0.42(m, 2H); ¹³C NMR (100 MHz, CDCl₃): 187.2, 151.2, 149.9, 137.3, 135.8,132.4, 131.1, 123.6, 55.8, 40.1, and 6.9; MS (ESI) [M+H]⁺ 318.5.

(3E,5E)-1-isopentyl-3,5-bis(2-methoxybenzylidene)piperidin-4-one (10-1)

Compound 10-1 was prepared as a hydrochloric acid salt (yellow powder).¹H NMR (400 MHz, CDCl₃): δ 13.19 (br, 1H), 8.36 (s, 2H), 7.45 (t, J=7.3Hz, 2H), 7.10 (d, J=6.6 Hz, 2H), 7.03 (t, J=7.3 Hz, 2H), 6.97 (d, J=8.3Hz, 2H), 4.49 (d, J=15.6 Hz, 2H), 4.28 (d, J=15.6 Hz, 2H), 3.88 (s, 6H),2.87-2.82 (m, 2H), 1.43 (septet, J=6.6 Hz, 1H), 1.30 (td, J=7.6, 6.6 Hz,2H), and 0.70 (d, J=6.6 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): 182.2, 158.2,141.4, 132.5, 130.5, 124.1, 122.2, 120.9, 111.3, 55.7, 50.8, 49.6, 32.8,26.0, and 22.1; MS (ESI) [M+H]⁺ 406.5.

(3E,5E)-1-isopentyl-3,5-bis(3-methoxybenzylidene)piperidin-4-one (10-2)

Compound 10-2 was prepared as a hydrochloric acid salt (yellow powder).¹H NMR (400 MHz, CDCl₃): δ 13.46 (br, 1H), 8.17 (s, 2H), 7.40 (t, J=8.0Hz, 2H), 7.01 (dd, J=8.0, 2.3 Hz, 2H), 6.89 (d, J=8.0 Hz, 2H), 6.86 (d,J=2.3 Hz, 2H), 4.59 (d, J=15.6 Hz, 2H), 4.48 (d, J=15.6 Hz, 2H), 3.84(s, 6H), 2.89-2.84 (m, 2H), 1.46 (septet, J=6.6 Hz, 1H), 1.37 (td,J=7.6, 6.6 Hz, 2H), and 0.72 (d, J=6.6 Hz, 6H); ¹³C NMR (100 MHz,CDCl₃): 181.8, 160.0, 144.8, 134.3, 130.4, 123.9, 122.2, 116.3, 116.1,55.5, 50.5, 50.2, 32.7, 26.0, and 22.1; MS (ESI) [M+H]⁺ 406.5.

Example 2: Cardiac Protection Promotion and Repair after MyocardialInfarction

The data herein show that administration of the compounds describedherein following myocardial infarction promotes cardiac protection andrepair. Among other functions, representative compounds prevented theincrease of infarct size, cardiac hypertrophy, and collagen deposition.The compounds significantly improved cardiac post-infarction function.The compounds also increased angiogenesis, promoted cardiac β-oxidation,and significantly decreased the levels of methylglutaryl carnitine, ametabolite associated with dilated cardiomyopathy.

Methods

All animal studies and protocols were approved by the InstitutionalAnimal Care and Use Committee at Baylor College of Medicine andconducted in strict accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals. Adult (8-10 week old)ICR (CD1) mice were used for all studies.

Model of heart failure in adult mice. To induce myocardial infarction(MI) in 8- to 10-week-old mice, the left anterior descending (LAD)artery was permanently ligated. Briefly, mice were anesthetized with 2%isoflurane and then intubated. The heart was exposed by performing athoracotomy through the fourth or fifth intercostal space and an 8-0nylon suture was tied around the LAD artery. 10 uls of Adenoviral SRC-3(Ad-SRC-3) or adenoviral GFP (Ad-GFP) at a titre of 9×10⁹ pfu/ml wasinjected into the anterior free wall near left ventricular descendingcoronary artery. To assess cell cycle entry, the analog5-ethynyl-2′-deoxyuridine (EdU; 0.2 g/L, Santa Cruz, SC-284628A) wasadded to the drinking water for the 9 day duration of the experiment.Cell proliferation was measured using Click-iT EdU kit (Invitrogen,C10339). The initial dose of MCB-613 was administered intraperitoneallyat 20 mg/kg 2 hours after surgery. Subsequent injections of the samedose were given for 6 additional days, and then repeated doses weregiven for 3 days at week 9 and week 16. Mice were harvested at indicatedtime points for analyses.

Echocardiography. Cardiac function was determined by echocardiography(VisualSonics, Vevo 2100, 40 Mhz-550S probe). After alignment in thetransverse B-mode with the papillary muscles, cardiac function wasmeasured on M-mode images.

Histological analysis. Whole hearts were fixed with 10% formalin,embedded in paraffin, and sectioned at 7-μm intervals. Each slide had 3or 10 sections, which started at the apex and ended at the sutureligation site (approximately 30-50 slides). Sections at the papillarylevel (slides 20-30) were stained with Picrosirius red to identify areasof fibrosis. Infarct size was determined using a length-based approach.

Immunostaining analysis. Immunohistochemistry and immunofluorescentstaining experiments were performed on FFPE (formalin-fixed andparaffin-embedded sections). TUNEL staining to detect apoptotic cellswas done using the DeadEnd™ Fluorometric TUNEL System manufacturer'sprotocol (Promega, GS3250).

Isolation of cardiac cells for single cell transcription profiling. Micewere placed under the surgical plane of anesthesia before cervicaldislocation. The hearts were removed and cells were isolated byLangendorff retrograde perfusion of calcium-free pH 7.4 Tyrodes solution(130 mM NaCl, 74.55 mM KCl, 0.5 mM MgCl, 0.33 mM NaH₂PO₄, 0.25 mm HEPES,22 mM glucose) with collagenase, 1 mg/mL for 15 minutes. The hearts werethen removed from the apparatus, and finely minced in the same Tyrodesbuffer with 15 mg/mL bovine serum albumin (BSA) before trituration witha glass pipette. Cardiomyocytes were then pelleted by differentialcentrifugation, 300 RPM for 3 minutes. The supernatant containing thenoncardiomyocyte population of cells was then filtered through a 70micron filter and pelleted at 750 G, and resuspended in 1.1 mL 2% fetalbovine serum (FBS) in phosphate buffered saline (PBS). 0.1 mL wasremoved for the “no stain control” for fluorescence-activated cellsorting (FACS). The other 1 mL was incubated with 4 ug/mL Calcein Blueand 10 microM DyeCycle Ruby and incubated at 37° C. for 10 minutes. Thecells were then spun down at 600G, and resuspended in 0.5 mL 2% FBS/PBScontaining Sytox Green (30 nM). Cells were then sorted for: SytoxGreen-, Calcein+, DyeCycle Ruby+ into 0.4% FBS in PBS using a FACS Ariaii cell sorter. The cells were then pelleted and resuspended in 100 uL0.4% FBS in PBS, counted, and then flowed through the 10× GenomicsChromium system for single cell transcriptomic profiling.

Metabolomic profiling. Hearts from 4 control and 4 MCB-613-treated micewere isolated 24 hours post-MI, perfused with 10 mM KCl, snap frozen inliquid nitrogen and stored at −80° C. 10 mgs of tissue was analyzed forfatty acids and carnitines normalized to 3 normal liver control samples.Fatty acids were normalized by Internal Standard L-Tryptophan andcarnitines normalized by Internal Standard L-Zeatine.

RER, VO₂ and VCO₂ measurements. Respiratory exchange ratio (RER), oxygenconsumption (VO₂) and carbon dioxide expiration (VCO₂) were measured byindirect calorimetry using a progressive maximal exercise test untilmice reached exhaustion.

Results

SRC-3 activation has low toxicity. Nuclear receptor coactivator 3(NCOA3) is expressed at low levels in human adult hearts, indicatingthat the activation of SRC-3 will have few side effects. In addition,MCB-613 has a low toxicity profile in vitro and in vivo. NCOA3expression in normal human hearts was determined and compared to muscletissues analyzed from the GTEx database from approximately 1,000autopsied donors. FIG. 1 contains graphs showing the expression of NCOA3in normal human hearts (left panel) and in muscle tissues (right panel).Fragments per kb gene length per million mapped reads (FPKM) are shownon the y-axis and the data points represent the samples. The data aresorted by level of expression of NCOA3.

SRC-3 expression induces the proliferation of non-cardiomyocytes inhearts. Adenovirus derived SRC-3 (Adeno-SRC3) was injected into the leftventricular free wall of a wild type adult mouse heart. Mice were fed5-ethynyl-2′-deoxyuridine-treated water (EdU water) as a cellproliferation marker. Hearts were harvested after nine days and stainedwith 4′,6-diamidino-2-phenylindole (Dapi) to identify nuclei, the PCM1cardiomyocyte marker, the cell surface marker wheat germ agglutinin(WGA), and the cell proliferation marker EdU. FIG. 2 is an image of theheart and shows that SRC-3 expression induces proliferation ofnon-cardiomyocytes in wild type adult mouse hearts.

The compounds described herein protect against early and progressiveloss of cardiac function after myocardial infarction. Mice were treatedwith representative compounds described herein after myocardialinfarction. FIG. 3A shows an experimental timeline for drug treatmentand echocardiography measurements after myocardial infarction. MCB-613or saline was administered two hours after ligation and for sixadditional days every 24 hours. Repeat injections were given three timesper week at weeks 8 and 16. The heart weight and tibia length weremeasured at week 10, as shown in FIG. 3B. Six mice were administeredsaline and seven mice were administered MCB-613. P<0.03. Leftventricular ejection fraction was determined by echocardiography (n=14up to 12 weeks; n=3 at 19 weeks) (FIG. 3C). Data were analyzed by ANOVAwith repeated measures and presented as means+/− SEM. P<0.001. FIG. 3Dcontains images of mouse hearts harvested after a myocardial infarctionand stained to visualize collagen fibers. Specifically, FIG. 3D showspicrosirius red stain of cross sections at the level of the papillarymuscle at 4x and corresponding 20× magnification of the infarct borderzone at 10 weeks post-myocardial infarction. Infarct size is presentedas % length. The data demonstrate a decrease in infarct size for theMCB-613 treated mice as compared to the control mice that wereadministered saline. Left ventricular ejection fraction was determinedby echocardiography for mice treated with Compound 10-1 after amyocardial infarction (see FIG. 4). FIG. 8 shows picrosirius red stainof cross sections at the level of the papillary muscle at six weekspost-myocardial infarction. Infarct size is presented as % length. Thedata demonstrate a decrease in infarct size for the Compound 10-1treated mice as compared to the control mice that were administeredsaline.

MCB-613 induces changes in major and minor non-cardiomyocyte cell typesin the heart post-myocardial infarction. Comprehensive single celltranscriptional profiling was performed of non-myocyte cells in theadult mouse heart. Single cell sequencing was performed onnon-cardiomyocytes in hearts at 10 weeks post-myocardial infarction.Cell clusters were generated by tSNE analysis and identified by geneexpression signatures. See FIG. 5A for a plot depicting a comprehensivesingle cell transcriptional profiling of non-myocyte cells in an adultmouse heart. FIG. 5B shows the different cell types present in anMCB-613 treated heart after myocardial infarction. FIG. 5C is a Vennanalysis of three cell clusters with endothelial signature, indicatingthat MCB-613 stimulates endothelial cell growth of two distinctendothelial cell populations in hearts at 10 weeks aftermyocardial-infarction.

MCB-613 increases cardioprotective carnitine metabolites. FIG. 6A is aheat map showing the metabolomics for long-chain fatty acids in mousehearts 24 hours after a myocardial infarction. FIG. 6B is a heat mapshowing the metabolomics for methylglutaryl carnitine in mouse heartsafter myocardial infarction (complete set FDR=1). The heat maps showthat MCB-613 increases cardioprotective carnitine metabolites, increasesβ-oxidation of long-chain fatty acids, and decreases methylglutarylcarnitine.

The compounds described herein stimulate SRC intrinsic transcriptionalactivity. HeLa cells transfected with a Gal4 responsive luciferasereporter (pG5-luc) and a construct encoding SRC-1, SRC-2 or SRC-3 fusedwith the DNA binding domain of Gal4 (pBIND-SRC-1, pBIND-SRC-2 orpBIND-SRC-3) were exposed to treatments with compounds as describedherein, including MCB-613, Compound 10-1, and Compound 10-2. FIG. 7,upper panel shows that MCB-613 selectively stimulates the intrinsictranscriptional activity of SRCs. FIG. 7, middle panel shows thatCompound 10-1 selectively stimulates the intrinsic transcriptionalactivity of SRCs. FIG. 7, bottom panel shows that Compound 10-2selectively stimulates the intrinsic transcriptional activity of SRCs.

Compound 10-1 improves cardiovascular fitness after myocardialinfarction. FIG. 9 contains graphs showing the results of a progressivemaximal exercise test in mice treated with saline (“saline”; n=3), micetreated with MCB-613 (“MCB-613”; n=3), and non-infarcted wild-type mice(“WT”; n=2). The upper panel shows the carbon dioxide expiration (VCO₂)and the lower panel shows the oxygen consumption (VO₂). As shown in FIG.9 through representative Compound 10-1, the compounds described hereinimproves cardiovascular and peripheral vascular fitness after myocardialinfarction.

SUMMARY

As shown by the data presented herein, the compounds described hereinstimulate angiogenesis in vivo and improve the function of injuredmyocardium. Thus, the compounds described herein are exceptionaltherapeutic agents that are useful in repairing and preventing chronicwounds, promoting angiogenesis, restoring blood flow in vasculardiseases, and constraining detrimental structural remodeling ofvulnerable myocardium by preventing metabolic remodeling. The compoundsare useful for cardiac repair after coronary insufficiency.

Example 3: Cardiac Protection Promotion and Repair after MyocardialInfarction Using Steroid Receptor Coactivator Stimulator

Progressive remodeling of cardiac tissue with loss of myocytes,inflammation, fibrosis and decrease in cardiac ejection fraction arehallmarks of myocardial infarction (MI)-induced heart failure. A keytherapeutic goal after MI is to protect the myocardium, minimize infarctsize, prevent progression to heart failure, and to support functionalrecovery. The data herein show that the small molecule stimulators ofsteroid receptor coactivators described herein promote new vessel growthand improve cardiac function following MI. As shown through arepresentative compound, administration of small molecule receptorcoactivator stimulators decreases infarct size, apoptosis, cardiachypertrophy, collagen deposition, and activates cardiomyocyte energeticpathways. Single-cell transcriptional profiling identified distinctinterstitial cell types and transcriptional responses associated withimproved cardiac function.

The compounds described herein represent novel therapeutic option forpreventing early and progressive loss of cardiac function after MI.

Methods

Animals. All animal studies and protocols were approved by theInstitutional Animal Care and Use Committee at Baylor College ofMedicine and conducted in strict accordance with the National Institutesof Health Guide for the Care and Use of Laboratory Animals. Adult (8-10week old) ICR (CD1) mice were used for all studies.

Angiogenesis assay for small molecule stimulator treatments. Five toseven day old specific-pathogen-free (SPF) certified fertilized chickeneggs (white leghorn) were used to access the chorioallantoic membrane(CAM). The total vessel area on the CAM was measured using imagesobtained prior to application of the drug. MCB-613 (100 μL) at aconcentration of 0.6 μM was topically applied onto the surface of theCAM. The vessel area on the CAM was monitored on a daily basis followingdaily application of the drug. Vehicle controls were maintainedthroughout the experiment. Treatments were continued for four days atthe end of which an image thresholding method (ImageJ) was used toquantify total vessel area on the CAM. Percent growth in vessel area wascompared for control and treated eggs. n=6 eggs per condition. Thisexperiment was repeated three times.

Angiogenesis Assay with drug treated mouse embryonic fibroblasts (MEFs).Five to seven day old SPF certified fertilized chicken eggs (whiteleghorn) were used to access the chorioallantoic membrane (CAM). Mouseembryonic fibroblasts (MEFs) were treated with 0.6 μM MCB-613 for 24hours. Post treatment, two million MEFs were suspended in 60 μL of PBScontaining magnesium and calcium and 40 μL of Matrigel (2M/egg) (CorningInc.; Corning, N.Y.). The MEFs were then mixed well by pipetting andtransferred to the surface of the CAM.

The vessel area on the CAM was monitored on a daily basis. Vehiclecontrols were maintained throughout the experiment. MEFs were allowed togrow on the CAM surface for four days at the end of which an imagethresholding method (ImageJ) was used to quantify total vessel area onthe CAM. Percent growth in vessel area was then compared across controland treated eggs (MEF treated versus untreated). n=6 eggs per condition.

Reporter assay. Cardiac fibroblasts were plated in six well plates andtransfected with a GAL4 responsive luciferase reporter (pG5-luc) andexpression vectors for a GAL4 DNA binding domain (GAL4-DBD) full lengthSRC-1, -2 or -3 fusion construct (pBIND-SRC-1, pBIND-SRC-2 orpBIND-SRC-3) or pBIND control using Lipofectamine 3000 (Invitrogen;Carlsbad, Calif.). Twenty-four (24) hours post transfection, cells weretreated with 6 M MCB-613 or dimethyl sulfoxide (DMSO) and incubatedovernight. Post treatment cells were lysed and total protein wasisolated using the Promega luciferase assay system (Promega LifeSciences; Madison, Wis.). Protein concentration was measured using aBradford assay (Bio-Rad Laboratories; Hercules, Calif.). Relative lightunits were measured and normalized to total protein concentration.

Model of heart failure in adult mice. To induce MI in eight- toten-week-old mice, the left anterior descending (LAD) artery waspermanently ligated. Briefly, mice were anesthetized with 2% isofluraneand then intubated. The heart was exposed by performing a thoracotomythrough the fourth or fifth intercostal space and an 8-0 nylon suturewas tied around the LAD. The initial dose of MCB-613 was administeredintraperitoneally at 20 mg/kg, two hours after surgery. Subsequentinjection at the same time of the day and same dose were given for sixadditional days, and then repeated doses were given for three days atweek nine and week 16. Mice were harvested at the indicated time pointsfor analyses.

Echocardiography. Cardiac function was determined by echocardiography(VisualSonics, Vevo 2100, 40 Mhz-550S probe). After alignment in thetransverse B-mode with the papillary muscles, cardiac function wasmeasured on M-mode images. Animal numbers for cardiac function in FIG.11A are Day 0 control (17); MCB-613 (15), Day 1 control (10); MCB-613(12), Day 14 vehicle (19); control (19), Day 56 control (12); MCB-613(15), Day 70 control (8); MCB-613 (10), Day 80 control (8); MCB-613(11), Day 133 control (3); and MCB-613 (3).

Histological analysis. Whole hearts were fixed with 10% formalin,embedded in paraffin, and sectioned at 7 m intervals. Each slide hadthree (3) to ten (10) sections, which started at the apex and ended atthe suture ligation site (approximately 30-50 slides). Sections at thepapillary level (slides 20-30) were stained with Picrosirius red toidentify areas of fibrosis.

Infarct size was determined using a length-based approach. TUNELstaining to detect apoptotic cells was performed using the DeadEnd™Fluorometric TUNEL System (Promega, GS3250).

Electron Microscopy. Animals were sacrificed and hearts were quicklyremoved and placed directly into cold primary fix (2%paraformaldehyde+2.5% glutaraldehyde+2 mM CaCl₂) in 0.1 M cacodylatebuffer, pH 7.4) where they were sliced in cross-section then held incold primary fix for four days. After fixation, tissues were stainedwith 0.1% tannic acid in 0.1 M cacodylate buffer, rinsed and osmicatedfor one hour, after which the tissue was rinsed in dH₂O andcounter-stained in aqueous uranyl acetate. Once again, the tissues wererinsed in dH₂O and then dehydrated in a gradient series of ethanol (50,70, 80, 90, 95, and 100%). Tissues were slowly infiltrated over a periodof four days with increasing dilutions of plastic resins to ethanol,respectively, until 100% plastic was reached. After a full day ofinfiltration in three changes of 100% plastic, the tissues were embeddedin freshly made Spurr's Low Viscosity resin and polymerized at 60° C.overnight. Ultra-thin sections of 55-65 nm were cut with a Diatome Ultra45 diamond knife, using a Leica UC7 ultramicrotome. Sections werecollected on 150 hex-mesh copper grids and viewed on a Hitachi H7500transmission electron microscope. Images were captured using an AMTXR-16 digital camera and AMT Image Capture, v602.600.51 software.

Isolation of cardiac cells. Mice were placed under the surgical plane ofanesthesia before cervical dislocation. The hearts were removed andcells were isolated by Langendorff retrograde perfusion of calcium-freepH 7.4 Tyrodes solution (130 mM NaCl, 74.55 mM KCl, 0.5 mM MgCl, 0.33 mMNaH2PO₄, 0.25 mm HEPES, 22 mM glucose) with collagenase, 1 mg/mL for 15minutes. The hearts were then removed from the apparatus, and finelyminced in the same Tyrodes buffer with 15 mg/mL BSA before triturationwith a glass pipette. Cardiomyocytes were then pelleted by differentialcentrifugation, 300 RPM for 3 minutes. The supernatant containing thenoncardiomyocyte population of cells was then filtered through a 70micron filter and pelleted at 750 g, and resuspended in 1.1 mL 2% fetalbovine serum (FBS) in phosphate buffered saline (PBS). Then, 0.1 mL ofthe mixture was removed for the “no stain control” forfluorescence-activated cell sorting (FACS). The other 1 mL of themixture was incubated with 4 μg/mL Calcein Blue and 10 μM DyeCycle Rubyand incubated at 37° C. for 10 minutes. The cells were then spun down at600 g, and resuspended in 0.5 mL 2% FBS/PBS containing Sytox Green (30nM). Cells were then sorted for: Sytox Green-, Calcein+, DyeCycle Ruby+into 0.4% FBS in PBS using a FACS Aria ii cell sorter. The cells werethen pelleted and resuspended in 100 μL 0.4% FBS in PBS, counted, andpassed through the 10× Genomics Chromium system.

Single-cell RNA-sequencing. Raw fastq files were imported into CellRanger 2.1.1 (10× Genomics) for alignment with STAR, filtering, barcodecounting, and UMI counting. To identify cell clusters and differentiallyexpressed genes, the Cell Ranger results were analyzed with Seurat suiteversion 3.0.0 implemented in R (version 3.4.3). Cells with <200or >5,000 unique genes expressed or >25% of reads mapping tomitochondria were removed as quality control measure. Filtered data wasnormalized and scaled within each sample and both wild type and treatedsamples were aligned with Seurat's alignment procedure for integratedanalysis. Wilcoxon rank sum test incorporated within Seurat was used toidentify differentially expressed genes across cell types or treatment.Gene ontology analysis was performed with a custom code developed inpython that utilizes hyper-geometric distribution to identify enrichedpathways (P-value <0.05).

To study the effect of treatment on cell-to-cell communication ininfarcted heart, curated and putative ligand-receptor pairs in humanwere obtained. For each cell type, a signature for drug treatment wasobtained by applying a filter of P-value<0.05 & log 2FC>0.25 or <−0.25.Cell-to-cell communication was built by linking cell types A and B,where ligand was differentially expressed in cell type A while receptoris differentially expressed in cell type B. The network was plottedusing the igraph R package.

Total RNA-Seq analysis. Sequencing reads were trimmed using thetrimGalore software. Next, the reads were mapped using HISAT against thehuman genome build UCSC mm10, and quantified using StringTie against theGencode gene model. Gene expression (FPKM) was quantile normalized usingthe R statistical system. Differentially expressed genes between tumorand normal samples were determined using a parametric t-test withp-value <0.05 and fold change of 1.25. Pathway enrichment analysis wascarried out using the GSEA software package; significance was achievedfor adjusted q-values (q<0.25). Heatmaps were generated using the usingMatplotlib, NumPy and SciPy libraries under python.

RNA isolation and qPCR. Total RNA was isolated from cells using theQiagen RNA isolation kit. cDNA was prepared with the VILO master mixreagent. qPCR analysis was carried out using the Taqman kit with primersfor Tlr7, Lcn2 and 18s.

Granulocyte isolation. Bone marrow cells were isolated from the rearlegs of mice treated with control or MCB-613 for 24 hours. Rear legswere removed and placed in ice-cold Hanks balanced salt solution (HBSS)(without Ca/Mg) plus 2% FBS. Both ends of the bone were cut and the bonemarrow was flushed with ice cold HBSS with 2% FBS using a 26G needle.Clumps were broken up with an 18G needle and filtered through a 70 μmfilter and centrifuged at 400 g for 10 min at 4° C. The pellet wassuspended in RBC lysis buffer (BD Biosciences Pharmigen; San Diego,Calif.) and incubated at room temperature for two minutes. HBSS buffer(8 mL) was added and spun at 400 g for 10 mins at 4° C. Viable cellswere counted by trypan blue exclusion and bone marrow granulocytes wereisolated using a mouse Neutrophil Isolation Kit from Miltenyi Biotec(Bergisch Gladbach, Germany).

Flow Cytometry and Cardiac Immune Phenotyping. Hearts and spleensisolated from control and MCB-613 treated mice 24 hours post-MI orpost-sham surgery were digested in digestion buffer: 500 μL of DNAse I(10 mg/ml), 500 μl Collagenase II (50 mg/ml) into 4 ml (enough forapprox. 12 spleens @ 1×) RPMI 1640. Cells were placed on a GentleMacsdissociator and run on “IMPC_step2” twice and incubated at 25° C. for 15minutes. The “IMPC_step2” program was repeated and the samples wereincubated at 25° C. an additional 15 minutes followed by another roundof the “IMPC_step2” program. Then, 400 μL of 4° C. Stopping Buffer (1xPBS, 0.1M EDTA) was added to each sample and centrifuged at ˜100 g forone second to collect liquid at the bottom of tube. Samples werefiltered through mesh filter caps into 50 mL conical tubes. The tubeswere then washed with 1 mL FACs buffer which was also then passedthrough the filter. Heart preps were more viscous and were washed with20 mL cold filtered saline. Samples were centrifuged at 500 g for 6minutes. Supernatant was discarded and the pellet suspended in 1 mL 4°C. FACS buffer. Following red blood cell (RBC) lysis and blocking,single cell suspensions were stained with an immune cell panel andquantified using an LSR II flow cytometer. Then, 500,000 live eventswere counted for spleen controls and the entire tube of heart cells wasrecorded and analyzed.

Western blots. Frozen whole hearts were pulverized using a mortar andpestle apparatus. Approximately 20 mg of powdered tissue was added to300 μL of radioimmunoprecipitation assay (RIPA) buffer and homogenizedusing a tissue homogenizer.

Samples were then incubated on a rotator platform at 4° C. for one hourfollowed by centrifugation at 12,000 g for 10 minutes to clear debris.Supernatants were collected and stored at −80° C. for future use.Protein concentration was determined using the bicinchoninic acid assay(BCA) reagent system. For cell lysates, NETN buffer with 10% glycerolwas used to lyse cells and isolate total protein. All lysis buffers weresupplemented with protease and phosphatase inhibitors. Tissue lysateprotein (30-50 μg) or cell lysate protein (50-70 μg) was loaded onto a4-15% gradient gel (Bio-Rad) and transferred onto a polyvinylidenedifluoride (PVDF) membrane. Immunoblotting was carried out usingantibodies for SRC-1, SRC-2, SRC-3, actin and Hsp90. HRP conjugatedanti-rabbit and anti-mouse secondary antibodies were used at dilutionsof 1:2,500. Pierce ECL was used for chemiluminescent detection.

Tube formation assay. Cardiac fibroblasts were treated with either DMSOor 6 μM MCB-613. Twenty four hours post treatment, drug was washed offby rinsing the cells twice with PBS. The cells were then conditionedwith endothelial cell growth media for 24 hours. Post conditioning,cells were plated on growth factor reduced matrigel (10 mg/ml) to allowfor tube formation overnight. The next day tubes were stained withCalcein AM and imaged using the Cytation imaging system.

Immunostaining. Hearts were perfused with cardioplegic 20 mM KCL-PBS andthen with 10% neutral buffered formalin before drop fixing and thenprocessing into paraffin wax. Sections (7 microns) were then cut andplaced onto slides. Immunofluorescence was performed by first removingthe paraffin and then rehydrating the sections. After that, antigenretrieval was performed (Antigen unmasking solution, Tris-based, VectorLabs cat #H-3301; Vector Labs, Burlingame, Calif.). Sections werepermeabilized with 0.1% tween20-PBS, blocked with 10% donkey serum in 1%tween20-PBS, and then incubated with primary antibody in blockingsolution (1:200 Rabbit anti-Lysozyme, abcam cat #AB108508; Abcam,Cambridge, United Kingdom), followed by secondary (1:200 Donkeyanti-Rabbit, Alexa 647, Thermo Fisher Scientific cat #A-31573; ThermoFisher Scientific, Waltham, Mass.), and then Rhodamine-conjugated WGA(1:250 Vector Labs Cat #RL-1022) and DAPI (1:500 Thermo FisherScientific Cat #62248). Images were taken on a Zeiss LSM780 confocalmicroscope. LYZ+ cells were counted manually from random images spanningthe entire myocardium of the left ventricle below the left anteriordescending coronary artery occlusion surgery. n=3 hearts/group, >10 mm²imaged/heart, 24 hours after MI surgery.

RER, VO₂ and VCO₂ measurements. RER, V02 and VCO₂ were measured byindirect calorimetry using a progressive maximal exercise test untilmice reached exhaustion.

Results

MCB-613 stimulates angiogenesis. The effects of MCB-613 on angiogenesisand stromal response, specifically in adult cardiac fibroblasts, wasstudied. SRC-1, 2, and 3 proteins were expressed in adult cardiacfibroblasts isolated from 10 week old mice (FIG. 10A). Cardiacfibroblasts were transfected with expression vectors for GAL4 DNAbinding domain-SRC-1, 2, and 3 fusion proteins and a GAL4-responsiveluciferase reporter to measure SRC activation following MCB-613treatment (FIG. 10B). SRC-3 activity was induced in response to MCB-613to a greater extent than for SRC-1 and SRC-2, indicating MCB-613preferentially stimulates SRC-3 activity in cardiac fibroblasts.Functionally, MCB-613 stimulated tube formation in adult cardiacfibroblasts in vitro (FIG. 10C). To investigate MCB-613's stimulation ofangiogenesis in vivo, a chick-egg angiogenesis assay was performed (FIG.10D). Administration of MCB-613 directly to chick eggs stimulatedangiogenesis in vivo. Additionally, introduction of mouse embryonicfibroblasts pre-stimulated with MCB-613 promoted robust angiogenesislikely in a cell non-autonomous manner. Not to be bound by theory, thesefindings indicate that MCB-613 stimulation of angiogenesis may occurthrough multiple mechanisms.

To determine whether MCB-613 ameliorates recovery after ischemia-inducedmyocardial injury, MCB-613 or vehicle control was administered to micetwo hours following myocardial injury induced by permanent surgicalligation of the left anterior descending coronary artery.

Increased angiogenesis was observed in the infarct border zone threedays post-MI indicating that MCB-613 promotes angiogenesis in injuredtissues and restores blood flow in the setting of vascular diseases(FIG. 10E).

MCB-613 prevents loss of cardiac function after MI. Surgical ligation ofthe mouse left anterior carotid artery is a commonly used pre-clinicalMI model for testing cardiovascular therapeutic interventions. Toexplore the role of SRC stimulation on early and late post-infarctioncardiac function and remodeling, mice that received MIs were treatedwith MCB-613 or vehicle control. Mice were given 20 mg/kg MCB-613 orcontrol vehicle by intraperitoneal injection two hours after MI surgeryand every 24 hours for six additional days (FIG. 11A). Early andprogressive loss of cardiac function after MI was measured byechocardiography prior to surgery, at 24 hours and at 2, 8, 12, and 19weeks post-surgery. Ejection fraction decreased to an average of 30% incontrol-treated animals 24 hours post-MI. In contrast, mice treated withMCB-613 two hours post-MI had an average ejection fraction of 43%,indicating MCB-613 prevented the early decrease in ejection fraction andprovided early protection to vulnerable myocardium (FIG. 11B). Ejectionfractions in control-treated mice decreased further after 24 hours andwas at its lowest 19 weeks post-MI, indicating progressive loss ofcardiac function overtime. In contrast, ejection fractions weremaintained above 40% from 24 hours post-MI until 19 weeks post-MIfollowing administration of MCB-613, indicating that the earlymyocardial protective effects of MCB-613 prevents progressive loss ofcardiac function. Repeat injections given for three days at weeks 8 and16 did not alter ejection fractions, indicating MCB-613 had no furtherimpact on cardiac function at later time points. Maintenance of cardiacfunction up to 19 weeks post-MI indicates that short-term earlyintervention may be effective in preventing congestive heart failureafter MI. Analysis of heart weights reveals that MCB-613 attenuated theMI-induced cardiomegaly compensatory response 12 weeks post-MI (FIG.11C), indicating that the preservation of cardiac function is correlatedwith prevention of another key feature of heart failure. Cardiacpositron emission tomography (PET) imaging was then used to make aspatial assessment of myocardial viability. Improved ¹⁸F-FDG uptake inthe infarct zone shows that MCB-613 preserves healthy myocardium 2 weekspost-MI (FIG. 11D). Heart tissue sections were stained with Picrosiriusred to evaluate infarct size and degree of fibrosis (FIG. 11E). Infarctsizes measured 12 weeks post-MI were larger in control-treated hearts(31% and 44%) compared to hearts from MCB-613-treated mice (3%, 14%, 20%and 22%). In addition, cardiomyocytes were smaller and associated withless fibrosis in the infarct border zone, demonstrating that MCB-613prevents two additional key molecular features of progressive heartfailure (FIG. 11E). Cardiac metabolic dysfunction is a common feature ofheart failure. SRCs coordinate diverse metabolic requirements in tissuesincluding in skeletal and heart muscle. Indirect calorimetry withexercise was performed to determine the impact of MCB-613 on energyexpenditure three weeks post-MI compared with age-matched mice withoutMI as a control (FIG. 12). VCO₂ and V02 are elevated in MCB-613 treatedanimals three weeks post-MI, indicating that MCB-613 can improve energyutilization during exercise in mice after MI. Thus, improved cardiacfunction is associated with improved energy expenditure. Electronmicrographs of hearts 72 hours post-MI shows that MCB-613 can preventdisorganization of myofibrillar structure and abnormal mitochondrialcristal architecture, indicating that MCB-613 protects cardiac muscleand mitochondria from MI induced damage (FIG. 11F). In support of earlymyocardial protection, MCB-613 prevents apoptosis 24 hours post-MI (FIG.11G). These findings indicate MCB-613 acts to directly preservefunctional myocardium and prevent detrimental remodeling of cardiactissue.

MCB-613 prevents cardiomyocyte damage response. To gain insight intocardiomyocyte- and non-cardiomyocyte-specific MCB-613 transcriptionalfunctions associated with mitigation of myocardial remodeling andimproved cardiac function, transcriptomic profiling was performed oncardiac cells purified from control-treated and MCB-613-treated mice 12weeks post-MI (FIG. 13A). Differential gene expression analysis ofcardiomyocytes indicates 122 upregulated genes and 107 downregulatedgenes are associated with improved cardiac function 12 weeks post-MI(FIG. 13B). Gene set enrichment analysis of differentially expressedgenes displayed a strong enrichment for gene onogeny categoriesrepresenting oxidative phosphorylation and adipogenesis and suppressionof apoptotic and inflammatory response (FIG. 13C), providing furthersupport that MCB-613 improves cardiac energy utilization in addition topreventing cardiomyocyte damage-associated signaling.

MCB-613 decreases inflammatory macrophages. Single cell transcriptomicprofiling was performed to identify cell types and cell-type-specificsignaling responses associated with improved cardiac function in MCB-613treated mice 12 weeks post-MI. Metabolically active, viable, single cellsuspensions of non-cardiomyocytes were prepared from whole heartsfollowing Langendorff perfusion (FIG. 13A). Minimal proceduralmanipulations were performed to prevent loss of cell types and tominimize the impact on transcriptional activity. Transcriptionalprofiles of 21,894 cells from two saline-treated mice and 21,474 cellsfrom two MCB-613 treated mice that passed RNA quality controls wereanalyzed using the 10× Chromium platform by Seurat analysis. Fifteendistinct cell clusters were identified based on cell expressionpatterns, unsupervised clustering and dimensional reduction analysisusing Seurat software analysis (FIG. 13D). Cluster sizes ranged from 101to 6,085 cells. Cell populations were identified based on known mousecardiac cell type markers (FIG. 13E). At 42% of total cells analyzed,macrophages are the major non-cardiomyocyte cell population 12 weekspost-MI, unlike in normal adult mouse hearts where non-cardiomyocytecells are comprised of only 10% hematopoietic-derived cells. Evaluationof the largest changes in cell number indicate macrophage cluster 1 andB lymphocytes decreased in number while epicardial cells, NK/Tlymphocytes, fibroblasts, and endothelial cells, including lymphaticcells, were increased in cell number in hearts from MCB-613 treated mice(FIG. 13F). Cardiac macrophages, fibroblasts, and endothelial cellpopulations display transcriptional heterogeneity and consist of four,three, and two sub-clusters, respectively. Evaluation of unique genesignatures in cardiac fibroblasts reveal the presence of aninjury-reactive fibroblast population expressing Postn (fibroblastcluster 2) and, in support of recently reported ‘homeostaticfibroblasts’ in remodeled hearts, fibroblasts in cluster 3 uniquelyexpress Comp (FIG. 14A). Genes uniquely enriched in fibroblast cluster 1indicate the presence of a fibroblast subpopulation involved insecretory functions that promote angiogenesis (Bmp4, Ecm1, Ccl11, Pgf)and extracellular matrix organization (Ecm2 and Pdgfra). Endothelialcell transcriptional signatures indicate the presence of 3sub-populations. Endothelial cluster 2 and lymphatic endothelial cellsexhibit increased transcriptional activation of 218 and 308 unique genesrespectively, compared to endothelial cluster 1, indicating divergentroles in cardiac maintenance 12 weeks post-MI (FIG. 14B). The lymphaticendothelial cluster is defined by unique expression of lymphaticendothelial genes Prox 1 and Lyve 1. Concurrent expression ofpro-angiogenic regulators Hif1a and Lrg1 and lymphangiogenic regulatorCc1121a, in addition to increased cell numbers, indicates MCB-613stimulates lymphangiogenesis 12 weeks post-MI. The gene expressionsignature in endothelial cluster 2, defined by unique expression ofearly cardiac genes Mkl2, Tek and Hand2 indicates a transcriptionalreversion to a more primitive cell state, likely due to an injury stressresponse. Not to be bound by theory, the largest endothelial cluster,endothelial cluster 1, represents endogenous homeostatic endothelialcells characterized by a small number of unique genes and the absence ofany associated GO-terms or signaling pathways. Transcriptionalsignatures for four macrophage subclusters were clearly separable (FIG.14C). Macrophage cluster 1, the largest subpopulation of macrophages, isdefined by expression of an inflammatory gene signature including Ccl8,Cc24, and Ly96 compatible with these genes' role in the resolution ofmyocardial inflammation. Cluster 2 represents a population of Ccr2+monocyte-derived macrophages that expresses inflammatory genes Cxcll,Ccr2, Ccr5 and Tlr2 that are known to be short-lived infiltratingmacrophages derived from bone marrow in response to injury. In contrast,macrophages in cluster 3 uniquely express 110 cell cycle proliferationgenes indicating the presence of a small population of proliferatingCcr2-macrophages known to be maintained by local proliferation that playa role in tissue repair and myogenesis. Macrophages in cluster 4 areidentified by the expression of genes involved in activation ofphagocytosis including Cd209 and Corola, indicating the presence of asmall population of phagocytic macrophages 12 weeks post-MI.Surprisingly, the rather small changes in cell population numbersassociated with improved cardiac function maintenance indicates thatMCB-613 cardioprotection is likely the result of cell functional changesinstead.

MCB-613 promotes salutary paracrine signaling. To determine interstitialcell-type functional responses associated with MCB-613 mediated improvedcardiac function 12 weeks post-MI, transcriptomic profiles were comparedin non-cardiomyocyte cells from control-treated and MCB-613-treated mice(FIG. 15A). Large variations in cell population transcriptionalresponses indicate MCB-613-selective cellular responses contribute toimproved cardiac function. Smaller populations of cells consisting oflymphatic and immune cell populations experienced the largestdrug-induced transcriptomic changes. To identify potential interstitialcell signaling interactions contributing to improved cardiac function,the number of interactions between ligands and receptors for each cellin control hearts compared to hearts from MCB-613 treated mice (FIG.15B) was calculated. The highest frequency of interactions occurredbetween ligands from each fibroblast population, one macrophage subtypeand the endothelial/SMC population broadcasting to granulocytereceptors. This pattern of interstitial cell signaling to granulocytesimplicates extensive paracrine regulation of granulocyte functions inthe MCB-613 cardio-protective response 12 weeks post-MI. Ligand-receptorpairings suggest coordinated regulation of tissue architecture andanti-inflammatory signaling pathways including MMP9-LRP1, HSP90B1-TLR7and SERPINE1-TAUR (FIG. 15C). In support of this, gene set enrichmentanalysis of up and downregulated gene signatures in granulocytesindicates MCB-613 suppresses inflammatory granulocyte functions (FIG.14D). The top up and down-regulated genes in cardiac granulocytes fromMCB-613-treated mice compared to control reveals increased expression ofgranules involved in innate defense and decreased cytokines, enzymes andchemokines involved in inflammatory signaling (FIG. 15D). These findingsindicate the myocardial response to MCB-613 is characterized by asustained paracrine anti-inflammatory signaling landscape that underliesimproved cardiac function.

Since administration of MCB-613 at the time of injury resulted in animmediate response at 24 hours (FIG. 11B and FIG. 1E), the effect ofMCB-613 on immune cells 24 hours post-MI was measured.Immune-phenotyping by FACS analysis of single cells isolated from wholehearts 24 hours post-MI was used to quantify the composition of immunecells. Similar to that seen at 12 weeks post-MI, there was a significantdecrease in B cells, a trend for decreased monocytes and no change inthe fraction of granulocytes in hearts from MCB-613 treated micecompared to controls (FIG. 16A). The acute granulocyte transcriptionalresponse to MCB-613 due to the presence of paracrine signaling and therobust transcriptional response in granulocytes 12 weeks post-MI wasthen measured. Granulocytes are the first innate immune cells to reachthe myocardium after acute ischemic injury and are critical mediators ofthe extent of the inflammatory reaction triggered by an acute heartattack and of the resulting damage to the heart muscle. Due to thedifficulty of isolating sufficient quantities of undamaged granulocytesfrom mouse hearts, to investigate granulocyte responses bone marrowgranulocytes were isolated, which reflect the myocardial granulocyteresponse 24 hours post-MI. Elevated mRNA expression of the granulocytemarker S100A9 in granulocytes compared to granulocyte-depleted bonemarrow indicates successful isolation of granulocytes (FIG. 16B).Increased expression of Tlr7 and Lcn2 in granulocytes from mice treatedwith MCB-613 supports the single cell transcriptomic analysis andreveals that modulation of granulocyte function may contribute to theacute myocardial response to MCB-613. To control for the possibilitythat MCB-613 regulates Tlr7 or Lcn2 in bone marrow granulocytes as aresult of tissue trauma from the surgical procedure in the absence of anMI, granulocytes were isolated from mice 24 hours following shamsurgeries with administration of control vehicle or MCB-613. No changesin cell numbers or gene expression were observed, indicating thatgranulocyte gene expression changes are a consequence of MCB-613mediated myocardial injury response. MCB-613 induction of a strongtranscriptome response in granulocytes indicates that neutrophilgranules can modulate the compound's post-MI inflammatory effects. Insupport of this, LYZ1 granule expression was significantly increased inthe myocardium of MCB-613 treated mice compared to control animals 24hours post-MI (FIG. 16C).

Example 4: Pharmacokinetic (PK) Studies of MCB-613, Compound 10-1, andCompound 10-2

The pharmacokinetics of MCB-613, Compound 10-1, and Compound 10-2 weretested in CD-1 mice. Each of the three compounds was dissolved in DMSO(20 mg/mL), mixed with 30% hydroxypropyl-o-cyclodextrin at a 1:9 ratio,and administered to the CD-1 mice intraperitoneally (ip) or orally (po)through a gavage. After compound administration, blood samples (3 miceper compound) were collected at nine time points, i.e., at 5 minutes,0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and24 hours, through the tail vein. Plasma was isolated from these bloodsamples and the compound plasma concentrations were determined byHPLC-MS/MS. Pharmacokinetic parameters were calculated using the programPKSolver, an add-in program for use in analyzing pharmacokinetic andpharmacodynamics data analysis, as described in Zhang et al., ComputerMethods and Programs in Biomedicine, 99: 306-314 (2010). The results aresummarized in Tables 1 and 2, and include the half-life (t/2), theterminal phase half-life (terminal phase t/2), the time afteradministration of the compound at which the maximum concentration isreached (t_(max)), the maximum concentration of the compound observed(C_(max)), the area under the curve up to the last measurableconcentration (AUC_(0-t)), the area under the curve to infinite time(AUC_(0-inf)), and the clearance rate of the compound (C₁).

Table 1 contains pharmacokinetic data from the above-described studiesin which MCB-613, Compound 10-1, and Compound 10-2 wereintraperitoneally administered to CD-1 mice.

TABLE 1 MCB-613 10-1 10-2 t_(1/2) (h) 0.86 0.64 0.25 Terminal phaset_(1/2) (h) 6.5 18.1 15.9 t_(max) (h) 0.08 0.25 0.08 C_(max) (ng/mL)49.2 356.7 463.3 AUC_(0-t) (ng/mL*h) 122.2 622.6 333.8 AUC_(0-inf)(ng/mL*h) 129.6 851.1 481.5 Cl (mg)/(ng/mL)/h 6.4 0.97 1.7

Table 2 contains pharmacokinetic data from the above-described studiesin which MCB-613, Compound 10-1, and Compound 10-2 were orallyadministered to CD-1 mice.

TABLE 2 MCB-613 10-1 10-2 t_(1/2) (h) 0.94 1.2 0.78 Terminal phaset_(1/2) (h) 16.1 10.2 27.1 t_(max) (h) 0.25 0.5 0.25 C_(max) (ng/mL) 5.653.2 181.9 AUC_(0-t) (ng/mL*h) 15.7 98.4 131.1 AUC_(0-inf) (ng/mL*h)24.3 112.6 134.6 Cl (mg)/(ng/mL)/h 34 7.3 6.1

The pharmacokinetic data are also illustrated in FIG. 17. The graphs inFIG. 17 show the average plasma concentration measured over time. Thetop row of graphs shows the pharmacokinetic data from theabove-described studies in which MCB-613 (top left graph), Compound 10-1(top middle graph), and Compound 10-2 (top right graph) wereintraperitoneally administered to CD-1 mice. The bottom row of graphsshows the pharmacokinetic data from the above-described studies in whichMCB-613 (bottom left graph), Compound 10-1 (bottom middle graph), andCompound 10-2 (bottom right graph) were orally administered to CD-1mice.

The compounds and methods of the appended claims are not limited inscope by the specific compounds and methods described herein, which areintended as illustrations of a few aspects of the claims and anycompounds and methods that are functionally equivalent are within thescope of this disclosure. Various modifications of the compounds andmethods in addition to those shown and described herein are intended tofall within the scope of the appended claims. Further, while onlycertain representative compounds, methods, and aspects of thesecompounds and methods are specifically described, other compounds andmethods are intended to fall within the scope of the appended claims.Thus, a combination of steps, elements, components, or constituents canbe explicitly mentioned herein; however, all other combinations ofsteps, elements, components, and constituents are included, even thoughnot explicitly stated.

1. A compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: A¹,A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are each independently selectedfrom CR¹ and N, wherein each R¹ is hydrogen, halogen, alkoxy, cyano,trifluoromethyl, or substituted or unsubstituted C₁₋₆ alkyl; and R² issubstituted or unsubstituted cycloalkyl or substituted or unsubstitutedheterocycloalkyl.
 2. The compound of claim 1, wherein the compound hasthe following formula:

wherein: m and n are each independently 1, 2, 3, 4, or
 5. 3. Thecompound of claim 1, wherein the compound has the following formula:

wherein: m and n are each independently 1, 2, 3, or
 4. 4. The compoundof claim 1, wherein the compound has the following formula:

wherein: m and n are each independently 1, 2, 3, or
 4. 5. The compoundof claim 1, wherein the compound has the following formula:

wherein: m and n are each independently 1, 2, 3, or
 4. 6. The compoundof claim 1, wherein R² is selected from the group consisting ofcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl.
 7. The compound of claim 1, wherein the compound is:


8. The compound of claim 1, wherein the compound is:

9-17. (canceled)
 18. A method for treating an ischemic injury in asubject, comprising: administering to the subject an effective amount ofa compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: A¹,A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are each independently selectedfrom CR¹ and N, wherein each R¹ is hydrogen, halogen, alkoxy, cyano,trifluoromethyl, or substituted or unsubstituted C₁₋₆ alkyl; and X isNR², CR³R⁴, or O, wherein R², R³, and R⁴ are each independently selectedfrom the group consisting of hydrogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocycloalkyl.
 19. The method of claim 18, wherein thecompound is selected from the group consisting of:


20. The method of claim 18, wherein the ischemic injury comprises amyocardial infarction or a stroke.
 21. The method of claim 18, furthercomprising selecting a subject who has suffered an ischemic injury,wherein the ischemic injury comprises a myocardial infarction or astroke.
 22. A method of reducing a myocardial infarct size, preventingor reducing cardiomyocyte loss, improving cardiac vascular perfusion,and/or improving central nervous system vascular perfusion in a subjectwho has suffered a myocardial infarction or stroke, comprising:administering to the subject an effective amount of a compound of thefollowing formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: A¹,A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are each independently selectedfrom CR¹ and N, wherein each R¹ is hydrogen, halogen, alkoxy, cyano,trifluoromethyl, or substituted or unsubstituted C₁₋₆ alkyl; and X isNR², CR³R⁴, or O, wherein R², R³, and R⁴ are each independently selectedfrom the group consisting of hydrogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocycloalkyl.
 23. The method of claim 22, wherein thecompound is selected from the group consisting of:


24. A method of improving cardiovascular function and/or central nervoussystem vascular function in a subject, promoting wound healing in asubject, or treating or preventing hypertrophic cardiomyopathy in asubject, comprising administering to the subject an effective amount ofa compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: A¹,A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are each independently selectedfrom CR¹ and N, wherein each R¹ is hydrogen, halogen, alkoxy, cyano,trifluoromethyl, or substituted or unsubstituted C₁₋₆ alkyl; and X isNR², CR³R⁴, or O, wherein R², R³, and R⁴ are each independently selectedfrom the group consisting of hydrogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocycloalkyl.
 25. The method of claim 24, wherein thecompound is selected from the group consisting of:


26. The method of claim 24, wherein the subject has suffered an ischemicinjury.
 27. The method of claim 26, wherein the ischemic injury is amyocardial infarction or a stroke.
 28. The method of claim 24, whereinthe subject is an elderly subject.